Method of identifying and treating premature infants at risk for BPD

ABSTRACT

Methods for identifying premature infants at risk for developing bronchopulmonary dysplasia and/or most likely to benefit from administration of inhaled nitric oxide for prevention of bronchopulmonary dysplasia (BPD). Methods for treating premature infants identified as at risk and/or likely to benefit are provided. also provided are methods for identifying premature infants that are not at risk for developing bronchopulmonary dysplasia and/or unlikely to benefit from administration of inhaled nitric oxide for prevention of bronchopulmonary dysplasia, and methods for avoiding risks of toxicity and undesirable side effects associated with inhaled nitric oxide therapy comprising administering only non-iNO treatment modalities to these infants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/320,635, filed Dec. 20, 2016 which is a National Phase entry ofInternational Application No. PCT/US2015/036413, filed Jun. 18, 2015,which claims priority to U.S. Provisional Application No. 62/014,817,filed Jun. 20, 2014, the disclosures of which are incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present invention relates to methods for identifying prematureinfants most 5 likely to benefit from administration of inhaled nitricoxide for prevention of bronchopulmonary dysplasia (BPD) and methods fortreating premature infants so identified.

BACKGROUND

INOMax®, (nitric oxide) (available from Ikaria, Inc. in Hampton, N.J.)for inhalation is an approved drug product for the treatment of term andnear-term (<34 weeks gestation) neonates having hypoxic respiratoryfailure associated with clinical or echocardiographic evidence ofpulmonary hypertension (INOmax label SPC-LBL 0303 R 9 hereinincorporated by reference).

Data from several animal models indicate that nitric oxide is requiredfor normal lung development. In vivo, nitric oxide is synthesized fromL-arginine by a family of nitric oxide synthase (NOS) enzymes. Theconstitutive isoenzymes endothelial nitric oxide synthase (eNOS) andneuronal nitric oxide synthase (nNOS) mediate nitric oxide in mammals. Acytokine-inducible isoform (iNOS) produces NO as an immune defensemechanism. It has been shown that mice genetically deficient in eNOS aresusceptible to mild neonatal hypoxia, with evidence of alveolarsimplification and a reduction in vascular volume that is not seen inwild-type mice. (Balasubramaniam et al. (2003) Amer. J. Physiol.—LungCellular and Molecular Physiology 284:L964-L971). Administration ofinhaled nitric oxide in mice with eNOS deficiency stimulated alveolarand vascular growth.

Survival among preterm infants has improved over time due toimprovements in the ability to enhance fetal lung maturation and tomanage respiratory distress syndrome. Chronic lung disease, i.e.,bronchopulmonary dysplasia (BPD), is now the most significant long-termpulmonary complication. BPD required prolonged hospitalization and isassociated with long-term pulmonary and neurodevelopmental problems. Inits more severe form, BPD is associated with inflammation, pulmonaryhypertension and increased airway resistance, and abnormalities in lungdevelopment.

Inhaled nitric oxide (iNO) has been used as a therapy for pulmonaryhypertension in full-term infants, but its efficacy for prevention ofBPD in premature infants is not clearly established. Published studiesreport both a benefit (i.e. decreased incidence of BPD) and a lack ofsuch benefit. These studies differ substantially in the preterm infantpatient populations studied, the treatment protocols, and determinationof outcome. Such mixed results suggest that the therapeutic response isvariable due variations in the patient population. The factorscontributing to this variability have not been identified and are notunderstood. There is therefore a need for methods for identifying asubpopulation of premature infants at risk for developing BPD that ismore likely to have a therapeutic response to iNO, as evidenced byprevention of long term pulmonary complications such as BPD. Identifyingthis subpopulation may also avoid administration of iNO to asubpopulation not likely to develop BPD. The present invention addressesthis need.

SUMMARY

INOMax®, (nitric oxide) for inhalation is an approved drug product.INOMax is a vasodilator, which, in conjunction with ventilator supportof other appropriate agents, is indicated for the treatment of term andnear-term (<34 weeks gestation) neonates with hypoxic respiratoryfailure associated with clinical or echocardiographic evidence ofpulmonary hypertension, where it improves oxygenation and reduces theneed for extracorporeal membrane oxygenation. The recommended dose ofINOmax for the approved indication is 20 ppm, maintained for up to 14days or until the underlying oxygen desaturation has resolved. Weaningshould occur gradually. Adverse reactions per the label includemethemoglobinemia and NO2 levels, both which can be dose dependent. Themost common adverse reaction is hypotension.

iNO may be administered via the INOMax DSIR®, INOmax® DS or INOvent®delivery systems which deliver operator-determined concentrations of NOin conjunction with a ventilator or breathing gas administration systemafter dilution with an oxygen/air mixture. A NO delivery system includesa NO administration apparatus, a nitrix oxide gas analyzer and anitrogen dioxide gas analyzer. Failure to calibrate the NO deliverysystem could result in under or over dosing of iNO. All delivery systemsshould be operated by a trained professional.

To ensure safe and effective administration of INOmax to avoid adverseevents associated with nitric oxide or NO₂, administration of INOmaxshould only be performed by a health care professional who has completedand maintained training on the safe and effective use of a Nitric OxideDelivery System provided by the manufacturer of the delivery system andthe drug.

INOmax is contraindicated in the treatment of neonates known to bedependent on right-to-left shunting of blood. Warning and precautionsincludes rebound pulmonary hypertension syndrome following abruptdiscontinuation. It is recommended to wean from INOmax as provided inthe label. Abrupt discontinuation of INOmax may lead to worseningoxygenation and increasing pulmonary artery pressure, i.e., ReboundPulmonary Hypertension Syndrome. Signs and symptoms of Rebound PulmonaryHypertension Syndrome include hypoxemia, systemic hypotension,bradycardia, and decreased cardiac output. If Rebound PulmonaryHypertension occurs, reinstate INOmax therapy immediately.

Hypoxemia from Methemoglobinemia should also be considered. Nitric oxidecombines with hemoglobin to form methemoglobin, which does not transportoxygen. Methemoglobin levels increase with the dose of INOmax; it cantake 8 hours or more before steady-state methemoglobin levels areattained. Monitor methemoglobin and adjust the dose of INOmax tooptimize oxygenation.

If methemoglobin levels do not resolve with decrease in dose ordiscontinuation of INOmax, additional therapy may be warranted to treatmethemoglobinemia (see INOmax label).

Additionally, nitrogen dioxide (NO₂) forms in gas mixtures containing NOand 02. Nitrogen dioxide may cause airway inflammation and damage tolung tissues. If the concentration of NO₂ in the breathing circuitexceeds 0.5 ppm, decrease the dose of INOmax. If there is an unexpectedchange in NO₂ concentration, when measured in the breathing circuit,then the delivery system should be assessed in accordance with theNitric Oxide Delivery System O&M Manual troubleshooting section, and theNO₂ analyzer should be recalibrated. The dose of INOmax and/or FiO₂should be adjusted as appropriate.

Other warning and precautions relation to heart failure. Patients withleft ventricular dysfunction treated with INOmax may experiencepulmonary edema, increased pulmonary capillary wedge pressure, worseningof left ventricular dysfunction, systemic hypotension, bradycardia andcardiac arrest. Discontinue INOmax while providing symptomatic care.

According to the INOmax label, no formal drug-interaction studies havebeen performed, and a clinically significant interaction with othermedications used in the treatment of hypoxic respiratory failure cannotbe excluded based on the available data. INOmax has been administeredwith dopamine, dobutamine, steroids, surfactant, and high-frequencyventilation. Although there are no study data to evaluate thepossibility, nitric oxide donor compounds, including sodiumnitroprusside and nitroglycerin, may have an additive effect with INOmaxon the risk of developing methemoglobinemia. An association betweenprilocaine and an increased risk of methemoglobinemia, particularly ininfants, has specifically been described in a literature case report.This risk is present whether the drugs are administered as oral,parenteral, or topical formulations.

Because clinical trials are conducted under widely varying conditions,adverse reaction rates observed in the clinical trials of a drug cannotbe directly compared to rates in the clinical trials of another drug andmay not reflect the rates observed in practice. The adverse reactioninformation from the clinical studies does, however, provide a basis foridentifying the adverse events that appear to be related to drug use andfor approximating rates.

Certain risk calculators may be used to assist in identifying if apatient is likely to develop BPD. Risk calculator software may take aninfant's gestational age, weight and ventilator support needs, amongother factors, into account. Such risk calculator may help a clinicianor therapist to assist in determining if a premature infant is at riskof developing BPD. The present invention identifies more concise andaccurate method for identifying if premature infant is at a risk ofdeveloping BPD and then administering iNO to a premature infant at saidrisk.

In one aspect the invention is directed to methods for identifyingpremature infants that are at risk for developing BPD and/or are likelyto have a therapeutic response to treatment with inhaled nitric oxide(iNO). The methods comprise measuring the concentration of a nitricoxide metabolite in the plasma of the infant prior to initiation of iNOtherapy, wherein a concentration of the metabolite that is lower than anormal concentration of the metabolite indicates that the infant is atrisk for developing BPD and/or is likely to have a therapeutic responseto treatment with iNO for prevention of BPD. In certain embodiments, thenitric oxide metabolite concentration is a concentration of nitrite,nitrate, or both. In further embodiments, a total concentration ofnitrite and nitrate of less than about 50 ?AM indicates that the infantis at risk for developing BPD and/or is likely to have a therapeuticresponse to treatment with iNO for prevention of BPD. This includes, incertain embodiments, instances in which the total nitrite and nitrateconcentration in the plasma is below the limits of detection in theassay. In a specific embodiment, a total concentration of nitrite andnitrate of about 0-45 μM, about 5-40 μM, about 10-30 [tM, about 201.1,M, or below the detection limit of the assay (i.e., effectively 0)indicates that the infant is at risk for developing BPD and/or is likelyto have a therapeutic response to treatment with iNO. In otherembodiments, a total concentration of nitrite and nitrate of at leastabout 50 viM indicates that the infant is not at risk for developing BPDand/or is unlikely to have a therapeutic response to treatment with iNO.In a specific embodiment, a total concentration of nitrite and nitrateof greater than about 60 pM, or about 60-185 μM, indicates that theinfant is not at risk for developing BPD and/or is unlikely to have atherapeutic response to treatment with iNO. Experimental data from aclinical trial show that the predictive accuracy for therapeuticefficacy of iNO administration is about 75% when iNO is administered toa premature infant at risk for BPD when the total plasma concentrationof nitrate and nitrite of the infant is less than about 50 μM, about0-45 μM, about 5-40 μM, about 10-30 μM, about 20 μM, or below thedetection limits of the assay. Experimental data from the clinical trialalso show that the predictive accuracy for lack of efficacy of iNOadministration is about 56% when the total plasma concentration ofnitrate and nitrite in the plasma of the premature infant is at leastabout 50 μM, at least about 60 μM, or about 60-185 μM.

In a second aspect the invention is directed to methods for identifyingpremature infants that are at risk for developing BPD and/or are likelyto have a therapeutic response to treatment with iNO, the methodscomprising measuring the concentration of nitric oxide (NO) in airexhaled by the infant prior to initiation of iNO therapy, wherein aconcentration of NO that is lower than a normal concentration of NO(i.e., lower than 2.5 ppb) indicates that the infant is at risk fordeveloping BPD and/or is likely to have a therapeutic response totreatment with iNO for prevention of BPD. In certain embodiments, aconcentration of NO in exhaled air that ranges from about 0.5-1.5 ppbindicates that the infant is at risk and/or is likely to have atherapeutic response to treatment with iNO. In a specific embodiment, aconcentration of NO in exhaled air that is about 1.5 ppb or less, about1 ppb or less, or ranges from about 0.5-0.9 ppb, indicates that theinfant is at risk and/or is likely to have a therapeutic response totreatment with iNO. In other embodiments, a concentration of NO in airexhaled by the infant prior to initiation of iNO therapy that is normalor higher than a normal concentration of NO (i.e., about 2.5 ppb orhigher) indicates that the infant is not at risk for developing BPDand/or is unlikely to have a therapeutic response to treatment with iNO.

In a third aspect the invention is directed to methods for identifyingpremature infants that are at risk for developing BPD and/or are likelyto have a therapeutic response to treatment with iNO, the methodscomprising obtaining a blood or tissue sample from the infant prior toinitiation of iNO therapy, and analyzing the blood, tissue, or othersample for a mutation in a gene encoding a nitric oxide synthase (eNOS,nNOS or iNOS) that results in decreased endogenous synthesis of nitricoxide, wherein the presence of the mutation indicates that the infant isat risk for developing BPD and/or is likely to have a therapeuticresponse to treatment with iNO. In certain embodiments, detection of aT-786C mutation or a GlU²⁹⁸→Asp polymorphism (G894T mutation) in theeNOS gene indicates that the infant is at risk and/or is likely to havea therapeutic response to treatment with iNO. In other embodiments,absence of a mutation in a gene encoding a nitric oxide synthase,indicating unimpaired enzyme activity and normal endogenous synthesis ofnitric oxide, indicates that the infant may not be at risk fordeveloping BPD and/or may be unlikely to have a therapeutic response totreatment with iNO.

In a further aspect the invention is directed to methods for treatingpremature infants at risk for developing BPD with iNO to prevent BPD,the methods comprising identifying a premature infant that is at riskfor developing BPD and is likely to have a therapeutic response totreatment with iNO using any of the foregoing identification methods,and administering iNO to the infant. In an alternative embodiment, theinvention is directed to methods for avoiding risks associated withadministration of iNO to premature infants at risk for developing BPD orfor prevention of BPD, the methods comprising identifying a prematureinfant that is not at risk for developing BPD and/or is unlikely to havea therapeutic response to treatment with iNO using any of the foregoingidentification methods, and treating the infant with one or moremodalities for prevention of BPD that do not include iNO. In certainembodiments, the premature infant that is at risk or not at risk fordeveloping BPD, and/or is likely or unlikely to have a therapeuticresponse to treatment with iNO is identified immediately after birth, onthe day of birth, before the third day after birth, or before the fifthday after birth. In certain embodiments the infant likely to have atherapeutic response is treated with iNO beginning at least five daysafter birth, or five to fourteen days after birth. In specificembodiments, treatment begins five to 10 days after birth, or 5 to 7days after birth. In further embodiments, treatment of a prematureinfant identified as at risk for developing BPD and/or is likely to havea therapeutic response to treatment with iNO comprises administeringdecreasing concentrations of NO, beginning at 20 ppm. In certainembodiments, the initial dose of 20 ppm NO is decreased to 10, 5 and 2ppm over the course of administration. In specific embodiments, theadministration of NO is for about 20-30 days, preferably a minimum of atleast 24 days. In a specific example of NO administration, the initialadministration of 20 ppm NO is for 48 to 96 hours, followed by 10 ppm 5,ppm and 2 ppm at weekly intervals.

DETAILED DESCRIPTION

Before describing several exemplary embodiments of the invention, it isto be understood that the invention is not limited to the details ofconstruction or process steps set forth in the following description.The invention is capable of other embodiments and of being practiced orbeing carried out in various ways.

It has been discovered that certain biomarkers present in prematureinfants are predictive of the risk of developing BPD and/or thelikelihood of a therapeutic response to iNO to prevent BPD. As usedherein, the term “therapeutic response to iNO” means that iNOadministration increases the rate of survival without bronchopulmonarydysplasia (BPD) at 36 weeks of post menstrual age compared to the rateof survival with development of BPD at 36 weeks of post menstrual age inan untreated premature infant population. As used herein, the term“premature infant”, “preterm infant” and the like means infants born atless than 30 weeks gestational age. The biomarkers identified are ofthree types: a) the concentration of metabolites of NO, such as nitriteand nitrate, in blood or plasma, b) the concentration of NO in exhaledair, and c) the presence or absence of a mutation in the eNOS gene thatdecreases endogenous NO production, or any combination thereof.

A first method for identifying premature infants that are at risk fordeveloping BPD and/or are likely to have a therapeutic response totreatment with iNO comprises measuring the concentration of one or morenitric oxide metabolites in the plasma of the infant prior to iNOtherapy, wherein a concentration of the metabolite that is lower than anormal concentration of the metabolite prior to treatment with iNOindicates that the infant is at risk and/or is likely to have atherapeutic response to treatment with iNO. A modification of the firstmethod may be used for identifying premature infants that are not atrisk for developing BPD and/or are unlikely to have a therapeuticresponse to treatment with iNO, so that non-iNO treatment modalities maybe administered and the risks of toxicity and undesirable side effectsof iNO therapy can be reduced. In this modification, if a concentrationof the metabolite is normal or higher than a normal concentration of themetabolite prior to treatment with iNO, iNO is not administered.Typically, in such cases only non-iNO treatment modalities will beadministered for prevention of BPD. By way of example, the metabolitethat is measured is nitrite and/or nitrate.

Several suitable tests are available for quantitating nitrite and/ornitrate in plasma. For example, the Nitrate/Nitrite Colorimetric AssayKit (Cayman Chemical Company, Ann Arbor, Mich.), the Nitrate/NitriteAssay (Kamiya Biomedical Company, Seattle, Wash.), and the Total NitricOxide and Nitrate/Nitrite Parameter Assay Kit (R&D Systems) are allsuitable for use in the invention as instructed by the manufacturer. Ithas been found that a total concentration of nitrite and nitrate inplasma of about 50 ?AM or more correlates with reduced risk ofdeveloping BPD and with non-responsiveness to iNO therapy. A totalconcentration of nitrite and nitrate of less than about 50 !AM,including concentrations that are below the limit of detection in theassay used (effectively 0), indicates that the premature infant is atrisk for developing BPD and/or is likely to have a therapeutic responseto treatment with iNO. In specific examples, the total concentration ofnitrate and nitrite from about 0-45 [tM, about 5-40 [tM, about 10-40[tM, about 10-30 1 AM, about 20 [tM, or below the detection limit of theassay indicates that the infant is at risk for developing BPD and/or islikely to have a therapeutic response to treatment with iNO. In specificexamples, a total concentration of nitrate and nitrite at least about 50RM, greater than about 60 [tM, or about 60-185 indicates that the infantis not at risk for developing BPD and/or is unlikely to have atherapeutic response to treatment with iNO.

A second method for identifying premature infants that are likely tohave a therapeutic response to iNO therapy comprises measuring theconcentration of NO in exhaled air while the infant breathes ambient airor air having all naturally-occurring NO removed. The measurement of NOconcentration in exhaled air is taken prior to treatment with iNO. Ifthe concentration of NO in air exhaled by the infant is lower than anormal concentration of exhaled NO, the infant is at risk for developingBPD and/or is likely to have a therapeutic response to iNO treatment forprevention of BPD. Typically, healthy pre-term infants exhale about 2.5ppb of NO when breathing ambient air. However, concentrations of NO inexhaled air from about 0.5-1.5 ppb indicate that the infant is at riskfor developing BPD and/or is likely to have a therapeutic response toiNO administration. In a particular embodiment a concentration ofexhaled NO that is about 1.5 ppb or less, about 1 ppb or less, forexample 0.5-0.9 ppb, indicates that the infant is at risk and/or islikely to have a therapeutic response to iNO administration. Amodification of the second method may be used for identifying prematureinfants that are not at risk for developing BPD and/or are unlikely tohave a therapeutic response to treatment with iNO, so that non-iNOtreatment modalities may be administered and the risks of toxicity andundesirable side effects of iNO therapy can be reduced. In thismodification, if a concentration of NO in exhaled air is normal orhigher than a normal concentration of NO prior to treatment with iNO(about 2.5 ppm or above), iNO is not administered. Typically, in suchcases only non-iNO treatment modalities for prevention of BPD will beadministered.

Several suitable instruments and devices are available for measuring theconcentration of NO in exhaled air, and can be adapted for use withpreterm infants. The analytical instrument should have a sensitivity forgas samples of at least 0.5 ppb and a range of at least 0.5-5 ppb. Forexample, exhaled breath samples may be analyzed using the Sievers NitricOxide Analyzer (NOA 280i) (GE Analytical Instruments, Boulder, Colo.).

A third method for identifying premature infants that are at risk fordeveloping BPD and/or are likely to have a therapeutic response to iNOadministration comprises, prior to initiation of iNO therapy, analyzinga blood, plasma, serum, orally component of blood, plasma or serum,tissue, or other sample, tracheal aspirates or the like, for thepresence of a mutation in a gene encoding a nitric oxide synthase (e.g.,eNOS, nNOS or iNOS), wherein the mutation results in decreasedendogenous synthesis of nitric oxide. Any known mutation or geneticanomaly in a nitric oxide synthase gene can be analyzed, provided itresults in decreased endogenous synthesis of nitric oxide. Examples ofmutations suitable for analysis include the T-786C mutation (a T-*Cmutation at position -786 in the 5′ flanking region of the eNOS gene)and G894T (a G-*T mutation at position 894 of the eNOS coding sequencethat results in a Glu²⁹⁸-*Asp polymorphism in the enzyme). Testing forthe T-786C mutation is commercially available through diagnosticlaboratories such as Molecular Diagnostics Laboratories (MDL, Covington,Ky.). In addition, any of the known laboratory methods for analysis ofsingle nucleotide polymorphisms (SNPs), insertions, deletions andgenotyping is suitable for use in the invention, e.g., melting curveanalysis of polymerase chain reaction products or polymerase chainreaction-restriction fragment length polymorphism analysis. Amodification of the third method may be used for identifying prematureinfants that are not at risk for developing BPD and/or are unlikely tohave a therapeutic response to treatment with iNO, so that non-iNOtreatment modalities may be administered and the risks of toxicity andundesirable side effects of iNO therapy can be reduced. In thismodification, if no mutation that reduced endogenous synthesis of nitricoxide is detected in a gene encoding a nitric oxide synthase, endogenoussynthesis of nitric oxide will be normal and iNO is not administered.Typically, in such cases only non-iNO treatment modalities forprevention of BPD will be administered.

The absence of a mutation in a gene encoding a nitric oxide synthasethat results in reduced endogenous synthesis of nitric oxide (indicatingnormal NOS activity) correlates with reduced risk of developing BPDand/or with a failure of therapeutic response to treatment with iNO forprevention of BPD. The presence of a mutation that reduces endogenoussynthesis of nitric oxide due to impairment of NOS activity indicatesthat the infant is at risk for developing BPD and/or is likely to have atherapeutic response to iNO treatment.

Any of the foregoing methods for identifying premature infants that areat risk for developing BPD and/or are likely to have a therapeuticresponse to iNO therapy for prevention of BPD can be used in connectionwith methods for preventing BPD in premature infants. In these methods,prior to initiation of iNO therapy, premature infants requiringmechanical ventilation are evaluated as discussed above forconcentrations of nitric oxide metabolites in their plasma,concentrations of exhaled nitric oxide while breathing ambient air orair from which substantially all naturally-occurring nitric oxide hasbeen removed, and/or a mutation in a gene encoding NOS that results indecreased endogenous synthesis of nitric oxide. If the result of one ormore of the tests indicates that the infant is at risk for developingBPD and is likely to have a therapeutic response to iNO administration,iNO therapy is initiated. Typically, if one or more of the testsindicates that the infant is not at risk and/or is unlikely to have atherapeutic response to iNO administration, administration of iNO willbe avoided and the infant may be treated only with one or more non-iNOmodalities for prevention of BPD. These identification tests provideimproved information for weighing the risk vs. benefit of treating theinfant with iNO, thereby reducing the risk of toxicity and undesirableside effects of the treatment. In addition, the identification testssubstantially increase the likelihood of efficacy of iNO treatment forthe individual premature infant.

Any treatment protocol known in the art for administering iNO topremature infants to prevent BPD is suitable for use in the presentinvention, after determining that the infant is at risk for developingBPD and/or is likely to have a therapeutic response to iNOadministration. Generally, one or more of the tests to identify thepremature infant that is at risk and/or is likely to have a therapeuticresponse to administration of iNO are performed from birth to about 14days after birth. In certain embodiments, the one or more tests areperformed between birth and about 5 days after birth. In specificembodiments, the one or more tests are performed immediately afterbirth, on the day of birth, before the third day after birth, or beforethe fifth day after birth.

It has been reported that on the third day after birth there is nodifference in exhaled NO in premature infants that subsequently developBPD and those that do not. Accordingly, particularly for evaluation ofexhaled NO, the evaluation is generally performed at a time betweenbirth and the third day after birth to obtain the most accurate results.

In general, administration of iNO to premature infants that have beenidentified as at risk for developing BPD and/or as likely to have atherapeutic response begins at least five days after birth. Treatment ofthese infants may begin 5 to 14 days after birth, 7 to 21 days afterbirth, or 5 to 7 days after birth. Administration of iNO generallycontinues for 20-30 days after it is initiated. In specific treatmentprotocols, administration of iNO may be continued for at least 24 days.

For example, the protocol disclosed by R. A. Ballard, et al. (2006. N.Eng. J. Med. 355:343-353) is a particularly useful treatment method. Inthis study, premature infants between 7 and 21 days of age requiringventilator support and at risk for developing BPD received decreasingconcentrations of nitric oxide, beginning at 20 ppm, for a minimum of 24days. The initial dose of nitric oxide was administered for 48-96 hours,and subsequent doses of 10, 5, and 2 ppm were administered at weeklyintervals thereafter. The authors report that administration of iNOaccording to this protocol improves the pulmonary outcome for prematureinfants who are at risk for BPD with no apparent short term adverseeffects.

A further example of a suitable administration protocol is that of M. D.Schreiber, et al. (2003. N. Engi. J. Med. 349:2099-2107), in whichpremature infants undergoing mechanical ventilation for respiratorydistress syndrome were treated with iNO at 10 ppm on day 1, followed by5 ppm for six days.

EXAMPLES

A multi-center, double blind, placebo-controlled randomized clinicaltrial was conducted to examine the efficacy of iNO in preterm infantsless than 30 weeks gestational age and less than 1250 grams who requiredmechanical ventilation or positive pressure support on days 5 to 14after birth. A secondary objective of the trial was to examine thepharmacokinetics (PK) of nitric oxide in preterm infants using plasmanitrite and nitrate (NOx) as surrogate biomarkers. Continuous iNOadministration was begun at 20 ppm by administration into theinspiratory limb of the ventilator circuit in the mechanicallyventilated subject using an INOvent® delivery device. Therapy wascontinued for 24 days, following a dose reduction schedule (10 ppm after72 hours of treatment, 5 ppm on day 10). If an infant was extubatedbefore 24 days, therapy was continued via nasal continuous positiveairway pressure or nasal cannula to complete the protocol.

Because of the challenge to measure free NO in systemic circulation, thePK assessment was done via plasma concentration data of NOx (nitrite andnitrate). In addition, methemoglobin (another metabolite of NO) was usedas a secondary variable for PK evaluation. This evaluation was done tounderstand the relationship between pharmacokinetic behavior of iNO andclinical outcomes. The sampling protocol included pre-treatment timepoints (less than 2 hours before initiation of NO dosing), as well as avariety of time points during iNO therapy and after discontinuation ofiNO therapy.

A liquid chromatographic/mass spectrophotometric (LC/MS) assay methodwas developed and qualified for measurement NOx in human plasma. Thequantitation of NOx was performed using the ¹⁵N-stable isotope labelednitrate as an internal standard. Nitrate reductase and NADH was added tothe samples for enzymatic reduction of nitrate to nitrite in plasma.Upon completion of the reduction reaction, acetone was added toprecipitate plasma proteins. The alkylating reagent, pentafluorobenzylbromide (PFBBr), was subsequently added to the samples to convertnitrite to a chemically stable pentafluorobenzyl derivative. Aftercompletion of derivatization, plasma supernatant was injected into theLC/MS for analysis. Plasma concentrations of total nitrite and nitratewere measured and reported as NOx using this assay method. Theinstrumental analysis of the nitrite and NOx samples was performed usingreversed-phase high performance liquid chromatography (HPLC), coupledwith mass spectrometric (MS) detection. A triple-quadrupole massspectrometer was used to acquire single-ion recording (SIR) data, andwas operated in the negative electrospray ionization mode for thedetection of the nitrite derivative (PFB—NO₂) and the ¹⁵N-labeledinternal standard derivative.

The NOx results for subjects that had a therapeutic response to iNOtreatment are shown in the following Table:

Subjects who had a response with Nitric Oxide Treatment Subject RaceTreatment Primary Lab Test Lab Test  425301 WHITE NITRIC Y <LLOQ uM 427801 WHITE NITRIC Y <LLOQ uM 1821202 BLACK NITRIC Y 77.5 uM 3412801WHITE NITRIC Y <LLOQ uM Mean NOx level 19.4 uMValues <LLOQ (below the limit of quantitation of the assay) were assumedto be zero (0)

The NOx results for subjects that did not have a therapeutic response toiNO treatment are shown in the following Table:

Subjects who DID NOT have a response with Nitric Oxide Subject RaceTreatment Primary Lab Test Lab Test  203801 HISPANI NITRIC N 62.3 uM 414501 WHITE NITRIC N <LLOQ uM 1110501 BLACK NITRIC N <LLOQ uM 1623801BLACK NITRIC N 184   uM 1816101 WHITE NITRIC N 62.5 uM 3631502 WHITENITRIC N <LLOQ uM 3632801 HISPANI NITRIC N <LLOQ uM 3635401 WHITE NITRICN 76.8 uM 3917501 WHITE NITRIC N 62.7 uM Mean NOx Level 49.8 uMValues <LLOQ (below the limit of quantitation of the assay) were assumedto be zero (0)

As can be seen from the results above, treatment with iNO was noteffective in subjects with NOx levels from 62.3 μM to 184 μM. Thecalculated mean NOx concentration for the non-responding population was49.8 μM. It can be concluded that a NOx concentration in plasma of about50 1 AM or above indicates that the subject is less likely to have atherapeutic response to iNO administration.

Conversely, the results show that treatment was therapeuticallyeffective in subjects having an average NOx concentration of about 20[NI or less. It can be concluded that a NOx concentration in plasma ofless than 50 μM, e.g., about 10-30 μM, indicates that the subject ismore likely to have a therapeutic response to iNO administration.

Reference throughout this specification to “one embodiment,” “certainembodiments,” “one or more embodiments” or “an embodiment” means that aparticular feature, structure, material, or characteristic described inconnection with the embodiment is included in at least one embodiment ofthe invention. Thus, the appearances of the phrases such as “in one ormore embodiments,” “in certain embodiments,” “in one embodiment” or “inan embodiment” in various places throughout this specification are notnecessarily referring to the same embodiment of the invention.Furthermore, the particular features, structures, materials, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It will be apparent to those skilled in the art thatvarious modifications and variations can be made to the method andapparatus of the present invention without departing from the spirit andscope of the invention. Thus, it is intended that the present inventioninclude modifications and variations that are within the scope of theappended claims and their equivalents.

What is claimed is:
 1. A method for identifying a premature infant thatis likely to have a therapeutic response to inhaled nitric oxidecomprising: a) prior to initiation of inhaled nitric oxide therapy,exposing the premature infant to ambient or nitric oxide free air, suchthat the premature infant inhales the air; b) obtaining a sample of theair upon exhalation; c) measuring a concentration of nitric oxide in theexhaled air; d) determining whether the measured concentration of nitricoxide is less than 1.5 ppb, wherein a concentration of less than about1.5 ppb indicates that the premature infant is likely to have areduction in the likelihood of developing bronchopulmonary dysplasiawhen treated with inhaled nitric oxide; and e) treating the prematureinfant with inhaled nitric oxide to reduce the likelihood of developmentof bronchopulmonary dysplasia when the measured concentration of nitricoxide in the exhaled air is less than about 1.5 ppb, and wherein thepremature infant is less than 30 weeks gestational age.
 2. The method ofclaim 1, wherein the measured concentration of nitric oxide is 0.5-1.5ppb.
 3. The method of claim 1, wherein the measured concentration ofnitric oxide is 1 ppb or less.
 4. The method of claim 2, wherein theconcentration of nitric oxide is measured between birth and the thirdday after birth.
 5. A method for reducing the likelihood of a prematureinfant at risk for of developing bronchopulmonary dysplasia, the methodcomprising: administering a dose of inhaled nitric oxide, wherein thepremature infant has a total concentration of nitrate and nitrite in theplasma that is less than about 50 μM, a concentration of nitric oxide inexhaled air that is less than about 1.5 ppb and/or a genetic mutation ina gene encoding nitric oxide synthase that results in decreaseendogenous synthesis of nitric oxide, wherein the premature infant isless than 30 weeks gestational age.
 6. The method of claim 5, comprisingdecreasing dosages of inhaled nitric oxide.
 7. The method of claim 6,comprising an initial dose of nitric oxide of 20 ppm for 48-96 hours,and subsequent doses of 10, 5 and 2 ppm at weekly intervals thereafter.8. The method of claim 5, which is initiated at least five days afterbirth.
 9. The method of claim 8, which is initiated at 5-14 days afterbirth.
 10. The method of claim 5, which is provided for 20-30 days.